Integrated analysis of how gender and body weight affect the intestinal microbial diversity of Gymnocypris chilianensis

Intestinal microorganisms that living in the mucosa and contents of the gastrointestinal tract of animals, have close links with their hosts over a long evolutionary history. The community structure of the fish intestinal microbiota is associated with food, living environment, and the growth stage. To screen for potential probiotics that can be used for regulating breeding behaviors, this study focused on the diversity of fish intestinal microorganisms. This study aimed to investigate the effects of sex and body weight on the intestinal microbial diversity of Gymnocypris chilianensis in the wild. The results showed that the significant high diversity and richness of intestinal microbiota were fould in heavier individuals, and males. The dominant bacterial phyla of G. chilianensis were Proteobacteria, Firmicutes, and Bacteroidetes. In addition, the abundance of Firmicutes varied significantly among different body weights. The genus profile revealed that small individuals were dominated by Weissella, while females were dominated by Aeromonas, and both large individuals and males were dominated by other genera. Phylogenetic relationships and UPGMA clustering analysis showed significant differences among the groups. In general, the two main factors that have an effect on the intestinal microbiota diversity of wild G. chilianensis are sex and body weight.

Intestinal microbial communities in G. chilianensis with different body weights and genders. The 29 samples were divided into two groups according to body weight; 19 fish weighing more than 300 g were considered large fish, and 10 fish weighing less than 300 g were considered small fish. G. chilianensis generally grow to about 300 g when it reaches sexual maturity (i.e., 4-year-old). In addition, the large 19 fish were divided into the male and female groups by their gonads. There were 10 fish in the male group which labeled by "M" in Table 1, and 9 fish in the female group which labeled by "F", respectively.
There were 10 dominant phyla across all samples, and these represented more than 98.41% of the entire sequence reads. Bacteria within the phylum Proteobacteria were dominant in the intestine of wild G. chilianensis with 51.01% of the population in the large fish and 45.47% in the small fish, respectively ( Fig. 2A). The content of Firmicutes in the small fish (41.52%) was significantly higher than that in the large fish (7.26%) (p < 0.05), so this phylum was selected as the most likely indicator species of the groups of large and small fish by random forest analysis (Fig. 2D).
From the perspective of gender grouping, Proteobacteria was the most represented bacteria in females and males (Fig. 2B). The content of Verrucomicrobia in the males (15.93%) was significantly higher than that in the females (3.73%) (p < 0.05). However, the content of Bacteroidetes in the females was significantly higher (p < 0.05)   www.nature.com/scientificreports/ with 10.01% in the females group, and 2.02% in the males groups. Besides, random forest analysis with the mean decrease Gini index found that Planctomycetes and Fusobacteria each served as indicator bacteria to distinguish between the two genders ( Fig. 2C).

Diversity of intestinal microbiota of G. chilianensis with different body weights and genders.
The Shannon index and Chao1 index of the large fish were higher than those of the small fish (Fig. 3A), thereby indicating that the species abundance and evenness degree of the intestinal microbiota of the large fish was higher than that of the small fish. In addition, the Shannon index of the male fish and Chao1 index of the female fish were higher in the sex comparison (Fig. 4A). The closer the samples were to each other, the more similar their microbiome structures were. Principal Component Analysis (PCA) based on the OTU abundance information of species found that samples within each group were closer to each other (Figs. 3B and 4B). Similarly, the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering analysis showed that the large fish, small fish, female fish, and male fish clustered in their respective branches within a small range (Figs. 3C and 4C). In all the samples, there were 8 genera with relative abundance greater than 1%, which were Aeromonas, Weissella, Luteolibacter, Cetobacterium, Eterobacter, Acinetobacter, Mycoplasma, Deefgea and Shewanella (Fig. 3C). Based on the relative abundance and difference analysis of the genera with average relative abundance greater than 1% in the sample, Luteolibacter was mainly enriched in the large fish, while Weissella and Cetobacterium were mainly enriched in the small fish (Fig. 3C). Additionally, the populations of G. chilianensis, Leuconostoc, Pseudomonas, Moellerella, Proteus, Ignatzschineria, Vagococcus, Akkermansia, and Aeromonas in the small fish were significantly higher than those in the large fish, while most of the other bacteria were significantly fewer in number in the small fish compared to the large fish ( Fig. 3D). For gender grouping, there were significant differences in two genera, namely Aeromonas in female fish and Luteolibacter in male fish, respectively (Fig. 4C). Besides, most bacteria (of Fusobacteria, Bacteroidetes, and Cyanobacteria) were significantly greater in number in the females than in the males (Fig. 4D).

The functional prediction of G. chilianensis intestinal microbiota differed by body weight and gender.
According to the interaction analysis of bacteria, most bacteria were related to each other. Firmicutes  5A). Functional analysis of the microbiota showed that the differentially expressed microbiota observed in G. chilianensis with different body weight and gender was mainly attributable to metabolic function. To further understand the differential changes in intestinal microbiota under different body weights and gender, we conducted a prediction of bacterial function and explored whether different body weights and gender would affect the function of intestinal microbiota. In the heavier fish, there were 20 upregulated pathways. The functional analysis of microbial communities in the two different gender groups showed that there were 3 upregulated pathways (cell motility, signal transduction and cellular community-prokaryotes) and 11 downregulated pathways (Metabolism of cofactors and vitamins, Metabolism of cofactors and vitamins, and Amino acid metabolism, among other pathways) in the female group (Fig. 5B).

Discussion
In terms of the Alpha diversity index, the Shannon index and the Simpson index of large individuals were significantly higher than those of small individuals, while those of male individuals were higher than female individuals. The results showed that the richness and evenness of intestinal microbiota increased with age and the flora became more stable. These results are consistent with those fingings for yellow catfish and grass carp 9,10 . The changes in the richness and evenness of intestinal microbiota may be caused by changes in the feeding habits of G. chilianensis as they age 12 . G. chilianensis is a kind of omnivorous fish with animal preference. In the larval stage, it mainly feeds on diatoms, cladocerans and other plankton. In the adult stage, it feeds more on benthos bait, and sometimes also eats young fish. In addition, the higher diversity index in large individuals may be related to strong swimming ability, but further research is needed to prove this. The differences in the intestinal microbiota between the sexes may be caused by the effects of sex-related hormones 13,14 .
Our study indicated that in the gut microbiome of G. chilianensis, the dominant flora of different genders and weights were relatively consistent, and included Proteobacteria, Firmicutes, and Bacteroidetes. At present, these three phyla have been dominant microbiome in the intestinal tracts of many marine and freshwater fish species [15][16][17] . The similar bacterial taxa in the intestinal microbiota of various fish species suggests that these bacteria are beneficial to the host and would contribute to digestion, nutrient absorption, and immune responses 4 . Noted that the Proteobacteria is the highest in relative abundance among these three phyla, belonging to the  18,19 . Bacteroidetes and Firmicutes are both highly important for the fermentation of polysaccharides, and there exists a mutually promoting symbiotic relationship between the two. They jointly promote the absorption and storage of energy in the host, and the change in their ratio consequently affects the metabolic potential of intestinal microbiota in the body. In this study, there were more Firmicutes and fewer Bacteroidetes observed in the intestines of the small fish, and these indicate the strong ability to decompose food and promote host energy absorption 20 . Bacterial interaction analysis also found that Firmicutes and Proteobacteria were associated with most bacteria, which was consistent with the results of previous studies 21,22 . At the genus level, the dominant bacterial groups of G. chilianensis with different weights and genders include Aeromonas, Weissella, and Enterobacter, which are all normal intestinal microbiota 23 . Aeromonas was the genus with the greatest variation in intestinal abundance among the different groups of G. chilianensis. They are widely distributed in nature, and some strains of this genus can efficiently produce amylase in fish to promote intestinal digestion 24 , while others may play a pathogenic role by causing enteritis, sepsis, and other diseases in both humans and animals 25 . In this study, the abundance of Aeromonas in the intestines of the small fish was higher than that of the large fish, and their abundance in females was higher than that of males. Whether such strains mainly promote nutrient absorption or cause disease in the body requires further study. Weissella are lactic acid bacteria that are ubiquitous in nature and can widely colonize the host intestinal tract 26,27 . The functions of Weissella including synthesize dextran, fructan, and other oligosaccharides. These functions of Weissella can promote the absorption of trace elements and reduce gastrointestinal discomfort, thus promoting the proliferation of Bifidobacterium 28 . The abundance of Weissella in the intestinal tract of small individuals was found to be significantly higher than that of large individuals, likely so that nutrients can be better absorbed to confer a www.nature.com/scientificreports/ faster growth rate 29 . The abundance of Enterobacter may be affected by the source of protein consumed 30 . Some bacteria of this genus are conducive to metabolic activities, while some are potential opportunistic pathogens 31 .
Whether there are differences in the abundance of Enterobacter in the gut caused by different protein sources in different sex individuals remains to be investigated 32 .
The phylogenetic relationships and UPGMA clustering analysis showed differences in the composition and structure of the intestinal microbiota between different body weights and genders, which was similar to those reported to be observed in aquatic animals such as Coreius guichenoti 33 . UPGMA clustering analysis showed that individuals with different body weights and sex clustered on their respective branches within a small range, which may be caused by the differences in the environment for different sampling sites within the same river 34 . The results of functional prediction showed that the intestinal microbiota functions of different body weights and genders were highly similar, and the dominant functions mainly included the metabolism of cofactors and vitamins, carbohydrate metabolism, and amino acid metabolism. However, there were some differences observed in the relative abundance of various functions between body weights and sex. Interestingly, Sugita et al. (1982) found that the normal intestinal microbiota of Mozambican tilapia (Oreochromis mossambicus) was established 20-60 days after hatching, and speculated that this establishment was related to the development of intestinal structure and function 35 . G. chilianensis primarily feed on other animals in the juvenile stage, while the adult fish are omnivorous 36 . Zooplankton, phytoplankton, and aquatic insects comprise the main sources of food. In order to adapt to this change in feeding habits, the intestinal microbiota of G. chilianensis concurrently changes in structure and function with the development of the digestive system.

Methods
Animal materials. All the test fish were collected in Shule River, Gansu Province, China, and the sampling site (96°48′18′′ E, 39°59′23′′ N) and growth environment were shown in Fig. 6. Fish samples were collected using gill nets (mesh: 5 × 5 cm) and ground bamboo cages (mesh: 1 × 1 cm) in April 2021. All fish were placed in oxygen-filled boxes, transported to the lab on ice, and measured for body length and weight. Fish samples that have reached sexual maturity were differentiated according to gonadal development, while fish samples that have not reached sexual maturity are unable to distinguish their gender. All were anesthetized with tricaine mesylate (MS-222) (Sigma-Aldrich, Beijing, China). The surface was sterilized with 75% ethanol before dissecting the entire intestine of the fish using sterile tools (scissors and tweezers). A similar weight (approximately 0.2 g) of intestinal contents was collected respectively from the foregut, midgut and hindgut of each fish and mixed into a single sample (approximately 0.6 g per fish). The individual intestinal contents were homogenized by brief vertexing.

Conclusion
In conclusion, our research provides the first detailed description of the microbiome structure of G. chilianensis under field conditions and demarcated between different genders and body weights. Alpha diversity analysis based on the metrics used in our study revealed that the large fish exhibited a higher intestinal microbiota richness and evenness compared to the small fish, which were also greater for males compared to females. The dominant bacterial groups of different genders and weights were relatively consistent. At the phylum level, Proteobacteria, Firmicutes and Bacteroidetes were dominant, while at the genus level, Aeromonas, Weissella, and Enterobacter were dominant. Both the variables of weight and sex had significant effects on microbial community structure. However, the clustering results showed that both sexes and weights clustered together in small areas, which may be caused by environmental differences in sampling locations. The difference between the different weights may be due to the ontogeny influence on the structure of the intestinal microbiota, and the difference between the sexes may be the result of the secretion of hormones. However, the results of this study should be treated with caution. It cannot be ruled out that environmental differences between sampling sites have an impact on intestinal microbiota structure. Future interspecific studies of individuals from different geographic areas and habitat types are needed to precisely define the role of these factors in shaping gut microbial composition. This study increases the understanding of the diversity of the fish intestinal microbial ecosystem, and provides both a scientific basis and theoretical guidance for the screening of intestinal probiotics to assist in the growth process and the study of probiotic preparations in the aquaculture industry.